CN110684118A

CN110684118A - T cell antigen coupler targeting CD19, expression vector and application thereof

Info

Publication number: CN110684118A
Application number: CN201910967259.6A
Authority: CN
Inventors: 张坤
Original assignee: Huaxia Yuan (shanghai) Cell Genetic Engineering Ltd By Share Ltd
Current assignee: Huaxia Yuan (shanghai) Cell Genetic Engineering Ltd By Share Ltd
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2020-01-14
Anticipated expiration: 2039-10-12
Also published as: CN110684118B

Abstract

The invention discloses a T cell antigen coupler targeting CD19, an expression vector and application thereof, and belongs to the field of tumor immunity drugs. The T cell antigen coupler targeting CD19, comprising: the polypeptide comprises a signal peptide, a single-chain antibody targeting CD19, a connecting sequence, a single-chain antibody targeting CD3, a mutated CD8 alpha hinge region, a CD8 alpha transmembrane region, an intracellular region, a P2A connecting peptide, an IL2 signal peptide and an anti-PD 1 single-chain antibody secretion region which are connected in sequence, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO: 1, the nucleotide sequence of the single-chain antibody targeting the CD19 is shown as SEQ ID NO: 2, the nucleotide sequence of the connecting sequence is shown as SEQ ID NO: 3, the nucleotide sequence of the mutated CD8 alpha hinge region is shown as SEQ ID NO: 4, respectively. The T cell antigen coupler targeting CD19 can reduce cytokine storm generated in the T cell treatment process, and simultaneously secrete single-chain antibody of anti-PD-1, thereby blocking PD-1: immunosuppressive signals between PD-L1/PD-L2.

Description

T cell antigen coupler targeting CD19, expression vector and application thereof

Technical Field

The invention relates to the field of tumor immunity drugs, in particular to a T cell antigen coupler targeting CD19, and an expression vector and application thereof.

Background

The malignant tumor is one of diseases seriously threatening human health, and according to incomplete statistics, the annual incidence rate of the malignant tumor in China currently reaches more than 400 ten thousand cases, wherein the incidence rate of blood tumors such as lymphoma, leukemia, multiple myeloma and the like is high. The Chimeric Antigen Receptor (CAR) technology is a cell therapy technology that has been developed very rapidly in recent years for treating malignant tumors. At present, although CAR-T cells can effectively kill tumor cells, they also bring adverse effects of cytokine storm, and can cause death of patients in severe cases. Therefore, there is a need for a safe T cell receptor for the treatment of malignancies.

Disclosure of Invention

In order to solve the problems in the prior art, the embodiment of the invention provides a T cell antigen coupler targeting CD19, an expression vector and application thereof. The technical scheme is as follows:

in one aspect, the invention provides a T cell antigen coupler targeting CD19, comprising: the polypeptide is characterized by comprising a signal peptide, a single-chain antibody targeting CD19, a connecting sequence, a single-chain antibody targeting CD3, a mutated CD8 alpha hinge region, a CD8 alpha transmembrane region, an intracellular region and an anti-PD 1 single-chain antibody secretion region which are connected in sequence, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO: 1, the nucleotide sequence of the single-chain antibody targeting CD19 is shown as SEQ ID NO: 2, the nucleotide sequence of the connecting sequence is shown as SEQ ID NO: 3, the nucleotide sequence of the mutated CD8 alpha hinge region is shown as SEQ ID NO: 4, the nucleotide sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO: 5, the nucleotide sequence of the intracellular region is shown as SEQ ID NO: 6, the nucleotide sequence of the P2A connecting peptide is shown as SEQ ID NO: 7, the nucleotide sequence of the IL2 signal peptide is shown as SEQ ID NO: 8, the nucleotide sequence of the anti-PD 1 single-chain antibody secretion region is shown as SEQ ID NO: shown at 9.

Specifically, the single chain antibody targeting CD3 is UCHT1, OKT3, L2K, or F6A.

Preferably, the CD 3-targeting single-chain antibody is UCHT1, and the nucleotide sequence of the CD 3-targeting single-chain antibody is shown in SEQ ID NO: shown at 10.

In another aspect, the present invention provides an expression vector comprising a vector and the T cell antigen coupler described above.

In particular, the vector is a lentiviral vector.

In another aspect, the present invention provides a use of the above-mentioned T cell antigen coupler targeting CD19, the use comprising: the T cell antigen coupler targeting CD19 is used as an anti-tumor drug.

In particular, the tumor comprises: acute lymphocytic leukemia, chronic B-lymphocytic leukemia, non-Hodgkin's lymphoma, and multiple myeloma.

The technical scheme provided by the embodiment of the invention has the following beneficial effects: the invention provides a T cell antigen coupler targeting CD19, the signal conduction of which is closer to the natural TCR complex and can reduce the cytokine storm generated in the T cell treatment process, and meanwhile, T cells constructed by the T cell antigen coupler (TAC) can play a stronger anti-tumor effect, thereby showing good application prospect. In addition, the T cell antigen coupler targeting CD19 can secrete anti-PD-1 single-chain antibodies, thereby blocking PD-1: immunosuppressive signaling between PD-L1/PD-L2, reducing T cell inhibitory signaling.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

Fig. 1 is a schematic structural diagram of a T cell antigen coupler targeting CD19 according to an embodiment of the present invention;

FIG. 2 is a graph of lentivirus transfection efficiency provided in example two of the present invention;

FIG. 3 is a graph comparing the secretion amounts of IFN γ, a cytokine provided in example two of the present invention, wherein T is a T cell not expressing TAC;

FIG. 4 is a graph comparing the secretion of the cytokine IL-2 provided in example two of the present invention, wherein T is a T cell which does not express TAC;

fig. 5 is a comparison graph of cell killing efficiency provided in the second embodiment of the present invention, where a is the killing ability of TAC-T cells to Raji cells constructed by the T cell antigen coupler targeting CD19 provided in the second embodiment of the present invention, B is the killing ability of T cells without TAC structures to Raji cells, the abscissa is the number ratio of effector cells to target cells, and the ordinate is cell killing efficiency in units%.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

Example one

The invention provides a T cell antigen coupler targeting CD19, comprising: a signal peptide (a human CD8 alpha signal region, a CD8 alpha leader), a single-chain antibody (aCD19) targeting CD19, a connecting sequence (L), a single-chain antibody targeting CD3, a mutated CD8 alpha hinge region (a human CD8 alpha hinge region in which the cysteines at the 164 th position and the 181 th position are mutated into serines), a CD8 alpha transmembrane region (a human CD8 alpha transmembrane region), an intracellular region, a P2A connecting peptide, an IL2 signal peptide and an anti-PD 1 single-chain antibody secretion region which are connected in sequence, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO: 1, the nucleotide sequence of the single-chain antibody targeting the CD19 is shown as SEQ ID NO: 2, the nucleotide sequence of the connecting sequence is shown as SEQ ID NO: 3, the nucleotide sequence of the mutated CD8 alpha hinge region is shown as SEQ ID NO: 4, the nucleotide sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO: 5, the nucleotide sequence of the intracellular region is shown as SEQ ID NO: 6, the nucleotide sequence of the P2A connecting peptide is shown as SEQ ID NO: 7, the nucleotide sequence of the IL2 signal peptide is shown as SEQ ID NO: 8, the nucleotide sequence of the anti-PD 1 single-chain antibody secretion region is shown as SEQ ID NO: shown at 9.

Specifically, the single chain antibody targeting CD3 may be UCHT1, OKT3, L2K, or F6A.

Preferably, the CD3 targeting single chain antibody may be UCHT1, and in this example, the nucleotide sequence of the CD3 targeting single chain antibody is shown in SEQ ID NO: shown at 10.

In this example, the anti-PD 1 single-chain antibody secretion region includes: heavy chain variable region V_HAnd light chain variable region V_L. Heavy chain variable region V_HAnd light chain variable region V_LAre connected through a flexible sequence, and the flexible sequence can be a connecting peptide (G)₄S)₃Specifically, the anti-PD 1 single-chain antibody secretion region has the structure of a heavy chain variable region V_HAnd a linker peptide (G)₄S)₃And light chain variable region V_L。

The sequence information of human CD8 alpha signal region, human CD8 alpha hinge region, human CD8 alpha transmembrane region and intracellular region gene is searched from NCBI website database, the clone number of the single-chain antibody (aCD3) targeting CD19 is FMC 63, and the heavy chain variable region V of the anti-PD 1 single-chain antibody secretion region (alpha PD1 scFV)_HAnd a linker peptide (G)₄S)₃And light chain variable region V_LThe sequence may be http: com/site, and is more suitable for human cell expression under the condition of ensuring that the coded amino acid sequence is not changed. The structure of the T cell antigen coupler targeting CD19 is shown in figure 1, the gene totally synthesizes the gene sequence of the T cell antigen coupler, the structure is CD8 alpha leader-aCD19-L-aCD3-CD8 alpha hinge-CD8 alpha transmembrane-CD8 alpha intra cellular-P2A-IL 2-alpha PD1, in figure 1, CD8 alpha hinge comprises a mutated CD8 alpha hinge region and a CD8 alpha transmembrane region, and the marker is CD 19-TAC.

The nucleotide sequence of the T cell antigen coupler is shown as SEQ ID NO: 11, and correspondingly, the amino acid sequence of the T cell antigen coupler is shown as SEQ ID NO: shown at 12.

Example two

The invention provides an expression vector, which comprises a vector and a T cell antigen coupler. The vector may be a lentiviral vector.

The preparation method of the expression vector is briefly described as follows:

carrying out PCR (Polymerase Chain Reaction) amplification to obtain a CD19-TAC gene sequence, respectively adding a restriction enzyme site Xba I and a restriction enzyme site EcoR I at two ends of the CD19-TAC gene sequence to obtain a substance to be restricted, and respectively carrying out double restriction enzyme Reaction of the Xba I and the EcoR I with a lentiviral vector plasmid pCDH-EF1-MCS-T2A-c opGFP to obtain a restriction enzyme fragment containing the CD19-TAC and a restriction enzyme fragment containing the pCDH-EF 1-MCS-T2A-copGFP. The enzyme digestion reaction conditions are as follows: the enzyme digestion temperature is 37 ℃ and the enzyme digestion time is 30 min. The enzyme system (total volume 50. mu.L) comprises: 5 μ L of 10 XBuf fer; 5. mu.g of DNA to be cleaved with an enzyme; 2 μ L of XbaI enzyme; 2 μ L of EcoRI enzyme; the volume of the digestion system was made up to 50. mu.L with deionized water.

Respectively carrying out electrophoresis on the enzyme-digested fragment containing CD19-TAC and the enzyme-digested fragment containing pCDH-EF1-MCS-T2A-copGFP by using agarose gel with the concentration of 1%, respectively cutting bands of the enzyme-digested fragment containing CD19-TAC and the enzyme-digested fragment containing pCDH-EF1-MCS-T2A-copGFP after the electrophoresis is finished, respectively putting the bands into two clean EP tubes, and then purifying and recovering DNA in the agarose gel to obtain a CD19-TAC enzyme-digested product and a pCDH-EF1-MCS-T2A-copGFP enzyme-digested product.

And connecting the obtained CD19-TAC enzyme digestion product with a pCDH-EF1-MCS-T2A-copGFP enzyme digestion product at 16 ℃ overnight to obtain a connection product pCDH-EF1-CD19TAC-T2A-copGFP, namely an expression vector. Wherein, the connection system with the total volume of 10 mu L comprises: mu.L of pCDH-EF1-MCS-T2A-copG FP restriction enzyme, 7. mu.L of CD19-TAC restriction enzyme, 1. mu.L of T4 DNA ligase and 1. mu.L of 10 XT 4 DNA ligase Buffer.

The ligation products were transferred to Stbl3 competent cells (purchased from TRANSGEN BIOTECH) as follows:

stbl3 competent cells stored in a-80 ℃ refrigerator were removed and thawed on ice. The ligation product was added to Stbl3 competent cells, ice-cooled for 30min, heat-shocked at 42 ℃ for 45s, and then ice-cooled for 2min to obtain the transformation product.

Adding the transformed product into 900 μ L liquid LB culture medium without antibiotic, fermenting and culturing at 37 deg.C with shaking table rotation speed of 200rpm for 45min to obtain fermentation liquid. The preparation method of the liquid LB culture medium without antibiotic addition comprises the following steps: 5g of imported yeast extract, 10g of imported peptone, 10g of anhydrous sodium chloride and 1L of sterile water are uniformly mixed, and the mixture is sterilized at 121 ℃ for 20min for use.

And centrifuging the fermentation liquor at 4000rpm for 5min, discarding the supernatant, retaining the precipitate, and resuspending the precipitate by adopting 100 mu L of liquid LB culture medium to obtain a resuspension solution.

The resuspended solution was applied to Amp-resistant solid LB plate medium (purchased from Komaga, Shanghai, microbial technologies, Ltd.), and the solid LB plate medium was placed in a bacterial incubator at 37 ℃ for overnight culture.

Positive clones were picked on solid LB plate medium.

The obtained positive clone is identified by the following specific method:

carrying out double enzyme digestion reaction on the obtained positive clone by Xba I and EcoR I, wherein the specific operation refers to the double enzyme digestion reaction of the substance to be digested, and carrying out agarose gel electrophoresis on the enzyme digestion product obtained by the positive clone to identify a target fragment so as to obtain the target fragment with the size of about 3000 bp. The target sequence is known to be a CD19-TAC gene sequence through sequencing identification.

And (3) plasmid extraction: and (3) preparing the positive clone with correct sequencing into an original bacterial liquid, inoculating the original bacterial liquid into 100mL of Amp resistant liquid LB culture medium, and carrying out overnight culture at 37 ℃ with the rotating speed of a shaking table of 200rpm to obtain the original bacterial fermentation liquid.

The original strain fermentation broth was centrifuged at 4000rpm for 10min, the supernatant was discarded, and the precipitate (thallus) was retained.

Plasmid of thallus is extracted by using an endotoxin-free plasmid large-scale extraction kit (purchased from Tiangen company), and the specific method is carried out according to the instruction of the kit.

Packaging of lentiviral vector plasmid: at about 8.5X 10 per dish 6h before transfection⁶Individual cells 293T cells were seeded into culture dishes of 10cm diameter. Ensure that the confluency of the cells is about 80% during transfection and the cells are uniformly distributed in a culture dish.

Preparing a solution A and a solution B, wherein the solution A comprises: 4mL of 2 XHEPES buffer (8 dishes packaged together), solution B comprising: 72ug of plasmid (target plasmid), 37.04ug of packaging plasmid PLP1, 34.8ug of packaging plasmid PLP2, 24.08ug of packaging plasmid PLP-VSVG, and 400 μ L of 2.5M calcium ion solution, the total volume of solution B being 4 mL. And fully mixing the solution B, slightly swirling the solution A, simultaneously dropwise adding the solution B into the solution A, and standing for 3-5 min to obtain a mixed solution. The mixture was vortexed gently, added dropwise to 293T cell-containing dishes, 1mL of the mixture was added to each dish, the dishes were gently shaken back and forth to distribute the mixture evenly on the surfaces of the dishes (care was taken not to rotate the dishes when shaking), and the dishes were placed in a 37 ℃ incubator for culture. After 12h of culture, the culture medium was replaced with fresh one and continued. After culturing for 48h, centrifuging the culture medium at 1500rpm/min for 5min, retaining the supernatant, collecting the supernatant containing the lentiviral vector plasmid, and filtering the supernatant with a filter membrane with the specification of 0.45 μm to obtain the filtrate containing the lentiviral vector plasmid.

The filtrate containing the lentiviral vector plasmid was transferred to an ultracentrifuge tube, and a 20% sucrose layer was carefully layered on the bottom of the ultracentrifuge tube (1 mL sucrose per 8mL filtrate containing the lentiviral vector plasmid). Equilibrating the ultracentrifuge tube with PBS (phosphate buffered saline), centrifuging at 27600rpm/min for 2h at 4 deg.C, carefully removing the ultracentrifuge tube, decanting the supernatant, inverting the ultracentrifuge tube to remove the residual supernatant and retaining the precipitate. Adding 150 μ LPBS into the ultracentrifuge tube, blowing and beating the bottom of the ultracentrifuge tube several times with a micropipette gun to dissolve the precipitate in PBS to obtain concentrated lentivirus (gene plasmid vector of T cell antigen coupler), subpackaging the concentrated lentivirus into the ultracentrifuge tube during implementation, and storing at-80 deg.C.

And (3) detecting the titer of the lentivirus: concentrated lentivirus was used to infect 293T cells (1X 10) at 0.5. mu.L, 5. mu.L and 50. mu.L, respectively⁵One/hole) for 24h, changing the liquid after 24h, extracting cell genome DNA after 72h, and diluting the genome DNA to 5-100 ng/mu L. Using TransLv^TMLentivirus qPCR transcription Kit (available from TransGen) was performed according to the instructions. The detection shows that the titer of the lentivirus is 3.6 multiplied by 10⁸TU/mL。

T cells (TAC-T) of a T cell antigen coupler are prepared by the following specific method:

preparation of PBMC (Peripheral Blood Mononulear Cell): 20mL of peripheral blood of the volunteer was collected, the peripheral blood was added to a 50mL centrifuge tube containing heparin, centrifuged at 2000rpm for 10min, and the upper plasma was transferred to a new centrifuge tube for cryopreservation. Adding 37 ℃ preheated normal saline with the same volume as the sediment into a centrifuge tube, fully and uniformly mixing, and carrying out blood cell sediment resuspension to obtain a resuspended cell sap. Another 50mL centrifuge tube was added with 20mL of the pre-warmed lymphocyte separation medium. 20mL of resuspended cell fluid was slowly added to the upper layer of lymphocyte separation fluid. Centrifuge at 800rpm for 20 min. And (3) sucking the upper plasma at a constant speed, stopping sucking the plasma when the plasma is 2-3 cm away from the leucocyte layer, quickly sucking the leucocyte layer cells, transferring the leucocyte layer cells into another new 50mL centrifuge tube, supplementing the volume of the centrifuge tube to 45mL by using physiological saline, centrifuging the centrifuge tube at 1200rpm for 5min, and repeating the process for 2 times for cleaning the cells. The cell pellet was resuspended using RPMI1640+ FBS medium at 10% concentration and the T cell number was calculated. In this example, the number of T cells was 1.2X 10⁷And (4) respectively.

Lentivirus transfection of human T cells: in this example, the T cell density was adjusted to 1X 10⁶and/mL, inoculating the T cells into anti-human 50ng/mL CD3 antibody and 50ng/mL CD28 antibody according to 1 mL/hole, adding 200IU/mL interleukin 2, and stimulating and culturing for 48 h. After two days of T cell activation, lentivirus was transfected with an infection rate of MOI 5, 8. mu.g/mL polybrene was added thereto, and the cells were cultured in an incubator at 37 ℃. And after 24h of transfection, replacing the culture medium, and continuously observing the growth condition of the cells, wherein the culture time is 8-13 days. Resulting in transfected CAR-T cells.

And (3) detecting the transfection efficiency of lentivirus: after transfection was complete, the transfected cells were observed periodically using an inverted fluorescence microscope. The transfected TAC-T cells were aspirated, the pellet was collected by centrifugation at 1000rpm for 5min, and the pellet was washed with a PBS solution. The proportion of cells expressing GFP fluorescence of transfected TAC-T cells was measured using the flow cytometer FITC channel. The transfection efficiency is shown in FIG. 2.

And (3) detecting the secretion of the cytokines of the TAC-T cells, which comprises the following steps:

in order to detect whether the TAC-T cells after lentivirus transfection are effectively activated, the TAC-T cells are co-cultured with T cells not expressing TAC respectively and then the secretion amounts of the cell factor IFN gamma and the cell factor IL-2 are detected by an ELISA kit. Specifically, each well will be 1 × 10, respectively⁶TAC-T cells and 1X 10 cells per well⁵Each target cell was seeded in 6-well plates at 37 ℃ with 5% CO₂And culturing for 24 h. The culture supernatant was aspirated, centrifuged at 1000rpm for 5min to remove cell pellet, and the culture supernatant was harvested. The culture supernatants were tested for the cytokine IFN γ and cytokine IL-2 according to the ELISA kit instructions. As shown in FIGS. 3 and 4, FIG. 3 is a graph showing a comparison between the secretion amounts of the cytokine IL-2 and FIG. 4 is a graph showing a comparison between the secretion amounts of the cytokine IFN γ. As can be seen from FIGS. 3 and 4, the secretion of both IFN γ and IL-2, which are cytokines, by TAC-T cells provided in the examples of the present invention, was increased, and it was found that the TAC-T cells transfected by lentivirus were effectively activated.

In vitro antitumor Effect of ① group-40 of the wells were placed in a 96-well plate and divided into eight groups of five wells, each group containing 200. mu.L of medium (denoted as Ab) alone in the first group and 100. mu.L of medium and 100. mu.L of 1X 10 in the second group⁴Target cells (denoted Ack), and a third group, to which 100. mu.L of medium and 100. mu.L of 1X 10 cells were added⁴One effector cell (designated Acn), 100. mu.L of 1X 10 cells were added to the fourth group⁴One target cell and 100. mu.L of 1X 10⁴Effector cells (As, effector cell count: target cell count 1: 1), and 100. mu.L of medium and 100. mu.L of 5X 10 cells were added to the fifth group⁴Each effector cell (designated Acn), 100. mu.L of 1X 10 cells were added to the sixth group⁴One target cell and 100. mu.L of 5X 10⁴Effector cells (As, effector cell count: target cell count 5: 1), and 100. mu.L of medium and 100. mu.L of 1X 10 cells were added to the seventh group⁵Each effector cell (designated Acn), in the eighth group was added 100. mu.L of 1X 10⁴One target cell and 100. mu.L of 1X 10⁵One effector cell (As, effector cell number: target cell number: 10: 1) ① group of target cells was Raji containing CD19 target protein, effectorThe cells are TAC-T cells transfected with TAC structures.

② group an additional 40 wells of the 96-well plate were divided into eight groups of five wells each, in the same manner as in group ①, and target cells of group ② were Raji containing CD19 target protein and effector cells were untransfected T cells.

After the 96-well plate is incubated for 4h, 20uL of CCK-8 solution is added to each well, the 96-well plate is incubated for 2h in an incubator, and the absorbance is measured at 450nm, cell killing efficiencies of ① groups and ② groups are calculated [1- (As-Acn)/(Ack-Ab) ] x 100% respectively according to the absorbance, wherein As is a test well (a culture medium containing target cells, effector cells and CCK-8), Ack is a target cell control well (a culture medium containing target cells and a CCK-8 solution), Acn is an effector cell control well (a culture medium containing effector cells, a CCK-8 solution), and Ab is a blank (a culture medium containing no cells and a CCK-8 solution), FIG. 5 is a comparison graph of cell efficiencies provided by the embodiment of the present invention, and As shown in FIG. 5, CD19TAC-T cells have significantly better killing ability to Raji cells than T cells without TAC structure, and thus, the expression vector provided by the embodiment of the present invention has good anti-tumor killing effect.

In another aspect, the present invention provides a use of the above T cell antigen coupler targeting CD19, the use comprising: the T cell antigen coupler targeting CD19 is used as an antitumor drug.

In particular, the tumor may comprise: acute lymphocytic leukemia, chronic B-lymphocytic leukemia, non-Hodgkin's lymphoma, and multiple myeloma.

The invention provides a T cell antigen coupler targeting CD19, the signal conduction of which is closer to the natural TCR complex and can reduce the cytokine storm generated in the T cell treatment process, and TAC (T cell antigen coupler) -T cells constructed by the T cell antigen coupler can play a stronger anti-tumor effect, thereby showing good application prospect. In addition, the T cell antigen coupler targeting CD19 is also able to reduce T cell inhibitory signals.

In practice, a single chain antibody targeting CD3 can be linked to an endogenous native T Cell Receptor (TCR), enabling the T cell antigen coupler to function closer to the native TCR complex. The connecting sequence is connected by an EAAAK repeated sequence, and the connecting sequence can form an alpha helix, can effectively separate two structural domains and form a relatively stable secondary structure in the middle, thereby reducing the possibility of being attacked by protease to a certain extent, which is very important for maintaining the functions of a single-chain antibody targeting CD19 and a single-chain antibody targeting CD 3. The mutated CD8 a hinge region enables more stable T cell antigen couplers targeting CD 19. The CD8 a transmembrane and intracellular regions enable anchoring of a T cell antigen coupler targeting CD19 on the T cell surface while providing a co-receptor domain. The anti-PD 1 single-chain antibody secretion region can block the combination of PD1 and PD-L1, block negative regulation signals, and block PD-1: immunosuppressive signals between PD-L1/PD-L2 reactivate T cells, thereby enhancing the immune response.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

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gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60

tcctgtgcag cttctggcta ctcctttacc ggctacacta tgaactgggt gcgtcaggcc 120

ccaggtaagg gcctggaatg ggttgcactg attaatcctt ataaaggtgt tagtacctat 180

aaccagaagt tcaaggaccg tttcactata agcgtagata aatccaaaaa cacagcctac 240

ctgcaaatga acagcctgcg tgctgaggac actgccgtct attattgtgc tagaagcgga 300

tactacggcg atagtgactg gtattttgac gtctggggtc aaggaaccct ggtcaccgtc 360

tcctcgggcg gcgggggttc tggtggcggc ggcagcggcg gtggaggatc agatatccag 420

atgacccagt ccccgagctc cctgtccgcc tctgtgggcg atagggtcac catcacctgc 480

cgtgccagtc aggacatccg taattatctg aactggtatc aacagaaacc aggaaaagct 540

ccgaaactac tgatttacta tacctcccgc ctggagtctg gagtcccttc tcgcttctct 600

ggttctggtt ctgggacgga ttacactctg accatcagca gtctgcaacc ggaagacttc 660

gcaacttatt actgtcagca aggtaatact ctgccgtgga cgttcggaca gggcaccaag 720

gtggagatca aacgaact 738

<210>11

<211>2736

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacta agttggaaat aacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420

ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480

ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540

cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600

tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660

gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720

cattattact acggtggtag ctatgctatg gactactggg gtcaaggaac ctcagtcacc 780

gtctcctcag aagccgccgc aaaggaagct gctgccaaag aagcagccgc taaggaggtt 840

cagctggtgg agtctggcgg tggcctggtg cagccagggg gctcactccg tttgtcctgt 900

gcagcttctg gctactcctt taccggctac actatgaact gggtgcgtca ggccccaggt 960

aagggcctgg aatgggttgc actgattaat ccttataaag gtgttagtac ctataaccag 1020

aagttcaagg accgtttcac tataagcgta gataaatcca aaaacacagc ctacctgcaa 1080

atgaacagcc tgcgtgctga ggacactgcc gtctattatt gtgctagaag cggatactac 1140

ggcgatagtg actggtattt tgacgtctgg ggtcaaggaa ccctggtcac cgtctcctcg 1200

ggcggcgggg gttctggtgg cggcggcagc ggcggtggag gatcagatat ccagatgacc 1260

cagtccccga gctccctgtc cgcctctgtg ggcgataggg tcaccatcac ctgccgtgcc 1320

agtcaggaca tccgtaatta tctgaactgg tatcaacaga aaccaggaaa agctccgaaa 1380

ctactgattt actatacctc ccgcctggag tctggagtcc cttctcgctt ctctggttct 1440

ggttctggga cggattacac tctgaccatc agcagtctgc aaccggaaga cttcgcaact 1500

tattactgtc agcaaggtaa tactctgccg tggacgttcg gacagggcac caaggtggag 1560

atcaaacgaa ctgcggccgc attcgtgccg gtcttcctgc cagcgaagcc caccacgacg 1620

ccagcgccgc gaccaccaac accggcgccc accatcgcgt cgcagcccct gtccctgcgc 1680

ccagaggcgt cccggccagc ggcggggggc gcagtgcaca cgagggggct ggacttcgcc 1740

tctgatatct acatctgggc gcccttggcc gggacttgtg gggtccttct cctgtcactg 1800

gttatcaccc tttactgcaa ccacaggaac cgaagacgtg tttgcaaatg tccccggcct 1860

gtggtcaaat cgggagacaa gcccagcctt tcggcgagat acgtcggaag cggagctact 1920

aacttcagcc tgctgaagca ggctggagac gtggaggaga accctggacc tatgtacagg 1980

atgcaactcc tgtcttgcat tgcactaagt cttgcacttg tcacaaacag tcaagtccaa 2040

ttggtggagt ctggcggtgg ggtagttcag cccggccgat ccctgcgcct ggactgcaaa 2100

gcttctggaa ttacgttctc aaactccgga atgcactggg tgcggcaagc acctgggaaa 2160

gggctggagt gggttgcggt gatttggtac gatggctcta agaggtacta cgcagacagc 2220

gttaaaggca gatttactat atcccgagat aactctaaaa atacgctctt cctccaaatg 2280

aatagcctga gggcagaaga cacagccgtt tactattgtg ctaccaatga tgattactgg 2340

ggacagggca ccctggttac cgtaagttcc ggcggtggtg gaagtggagg agggggatcc 2400

ggaggcgggg gttctgagat cgtcctgacc cagtctccag ccactctctc cctgtctcca 2460

ggcgagcgcg ctacactgag ttgtagagct tcccagtccg tgagcagcta tctggcctgg 2520

tatcagcaga agcctgggca ggctccacgg ttgctgattt atgacgcctc caaccgcgcg 2580

actgggatac cagctaggtt ttccggatca ggcagcggca ctgattttac actgaccatc 2640

tcatctctcg agccggaaga tttcgccgtt tactattgtc aacagagttc aaactggcca 2700

cggacattcg gtcaggggac caaggttgaa attaag 2736

<210>12

<211>912

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>12

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Ala Pro Ala Ile Gly Met Thr Gly Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Ala Ala Val Thr Ile Ser Cys Ala Ala Ser Gly

35 40 45

Ala Ile Ser Leu Thr Leu Ala Thr Thr Gly Gly Leu Pro Ala Gly Thr

50 55 60

Val Leu Leu Leu Ile Thr His Thr Ser Ala Leu His Ser Gly Val Pro

65 70 75 80

Ser Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Thr Ser Leu Thr Ile

85 90 95

Ser Ala Leu Gly Gly Gly Ala Ile Ala Thr Thr Pro Cys Gly Gly Gly

100 105 110

Ala Thr Leu Pro Thr Thr Pro Gly Gly Gly Thr Leu Leu Gly Ile Thr

115 120 125

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Val Leu Leu Gly Gly Ser Gly Pro Gly Leu Val Ala Pro Ser Gly Ser

145 150 155 160

Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Ala Thr Gly

165 170 175

Val Ser Thr Ile Ala Gly Pro Pro Ala Leu Gly Leu Gly Thr Leu Gly

180 185 190

Val Ile Thr Gly Ser Gly Thr Thr Thr Thr Ala Ser Ala Leu Leu Ser

195 200 205

Ala Leu Thr Ile Ile Leu Ala Ala Ser Leu Ser Gly Val Pro Leu Leu

210215 220

Met Ala Ser Leu Gly Thr Ala Ala Thr Ala Ile Thr Thr Cys Ala Leu

225 230 235 240

His Thr Thr Thr Gly Gly Ser Thr Ala Met Ala Thr Thr Gly Gly Gly

245 250 255

Thr Ser Val Thr Val Ser Ser Gly Ala Ala Ala Leu Gly Ala Ala Ala

260 265 270

Leu Gly Ala Ala Ala Leu Gly Val Gly Leu Val Gly Ser Gly Gly Gly

275 280 285

Leu Val Gly Pro Gly Gly Ser Leu Ala Leu Ser Cys Ala Ala Ser Gly

290 295 300

Thr Ser Pro Thr Gly Thr Thr Met Ala Thr Val Ala Gly Ala Pro Gly

305 310 315 320

Leu Gly Leu Gly Thr Val Ala Leu Ile Ala Pro Thr Leu Gly Val Ser

325 330 335

Thr Thr Ala Gly Leu Pro Leu Ala Ala Pro Thr Ile Ser Val Ala Leu

340 345 350

Ser Leu Ala Thr Ala Thr Leu Gly Met Ala Ser Leu Ala Ala Gly Ala

355 360 365

Thr Ala Val Thr Thr Cys Ala Ala Ser Gly Thr Thr Gly Ala Ser Ala

370375 380

Thr Thr Pro Ala Val Thr Gly Gly Gly Thr Leu Val Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

405 410 415

Ile Gly Met Thr Gly Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Ala

420 425 430

Ala Val Thr Ile Thr Cys Ala Ala Ser Gly Ala Ile Ala Ala Thr Leu

435 440 445

Ala Thr Thr Gly Gly Leu Pro Gly Leu Ala Pro Leu Leu Leu Ile Thr

450 455 460

Thr Thr Ser Ala Leu Gly Ser Gly Val Pro Ser Ala Pro Ser Gly Ser

465 470 475 480

Gly Ser Gly Thr Ala Thr Thr Leu Thr Ile Ser Ser Leu Gly Pro Gly

485 490 495

Ala Pro Ala Thr Thr Thr Cys Gly Gly Gly Ala Thr Leu Pro Thr Thr

500 505 510

Pro Gly Gly Gly Thr Leu Val Gly Ile Leu Ala Thr Ala Ala Ala Pro

515 520 525

Val Pro Val Pro Leu Pro Ala Leu Pro Thr Thr Thr Pro Ala Pro Ala

530535 540

Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gly Pro Leu Ser Leu Ala

545 550 555 560

Pro Gly Ala Ser Ala Pro Ala Ala Gly Gly Ala Val His Thr Ala Gly

565 570 575

Leu Ala Pro Ala Ser Ala Ile Thr Ile Thr Ala Pro Leu Ala Gly Thr

580 585 590

Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Thr Cys Ala His

595 600 605

Ala Ala Ala Ala Ala Val Cys Leu Cys Pro Ala Pro Val Val Leu Ser

610 615 620

Gly Ala Leu Pro Ser Leu Ser Ala Ala Thr Val Gly Ser Gly Ala Thr

625 630 635 640

Ala Pro Ser Leu Leu Leu Gly Ala Gly Ala Val Gly Gly Ala Pro Gly

645 650 655

Pro Met Thr Ala Met Gly Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala

660 665 670

Leu Val Thr Ala Ser Gly Val Gly Leu Val Gly Ser Gly Gly Gly Val

675 680 685

Val Gly Pro Gly Ala Ser Leu Ala Leu Ala Cys Leu Ala Ser Gly Ile

690 695700

Thr Pro Ser Ala Ser Gly Met His Thr Val Ala Gly Ala Pro Gly Leu

705 710 715 720

Gly Leu Gly Thr Val Ala Val Ile Thr Thr Ala Gly Ser Leu Ala Thr

725 730 735

Thr Ala Ala Ser Val Leu Gly Ala Pro Thr Ile Ser Ala Ala Ala Ser

740 745 750

Leu Ala Thr Leu Pro Leu Gly Met Ala Ser Leu Ala Ala Gly Ala Thr

755 760 765

Ala Val Thr Thr Cys Ala Thr Ala Ala Ala Thr Thr Gly Gly Gly Thr

770 775 780

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

785 790 795 800

Gly Gly Gly Gly Ser Gly Ile Val Leu Thr Gly Ser Pro Ala Thr Leu

805 810 815

Ser Leu Ser Pro Gly Gly Ala Ala Thr Leu Ser Cys Ala Ala Ser Gly

820 825 830

Ser Val Ser Ser Thr Leu Ala Thr Thr Gly Gly Leu Pro Gly Gly Ala

835 840 845

Pro Ala Leu Leu Ile Thr Ala Ala Ser Ala Ala Ala Thr Gly Ile Pro

850 855860

Ala Ala Pro Ser Gly Ser Gly Ser Gly Thr Ala Pro Thr Leu Thr Ile

865 870 875 880

Ser Ser Leu Gly Pro Gly Ala Pro Ala Val Thr Thr Cys Gly Gly Ser

885 890 895

Ser Ala Thr Pro Ala Thr Pro Gly Gly Gly Thr Leu Val Gly Ile Leu

900 905 910

Claims

1. A T-cell antigen coupler targeting CD19, comprising: the anti-PD 1 single-chain antibody comprises a signal peptide, a single-chain antibody targeting CD19, a connecting sequence, a single-chain antibody targeting CD3, a mutated CD8 alpha hinge region, a CD8 alpha transmembrane region, an intracellular region, a P2A connecting peptide, an IL2 signal peptide and an anti-PD 1 single-chain antibody secretion region which are connected in sequence, wherein the nucleotide sequence of the signal peptide is shown as SEQ ID NO: 1, the nucleotide sequence of the single-chain antibody targeting CD19 is shown as SEQ ID NO: 2, the nucleotide sequence of the connecting sequence is shown as SEQ ID NO: 3, the nucleotide sequence of the mutated CD8 alpha hinge region is shown as SEQ ID NO: 4, the nucleotide sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO: 5, the nucleotide sequence of the intracellular region is shown as SEQ ID NO: 6, the nucleotide sequence of the P2A connecting peptide is shown as SEQ ID NO: 7, the nucleotide sequence of the IL2 signal peptide is shown as SEQ ID NO: 8, the nucleotide sequence of the anti-PD 1 single-chain antibody secretion region is shown as SEQ ID NO: shown at 9.

2. The T-cell antigen coupler of claim 1, wherein the single chain antibody targeting CD3 is UCHT1, OKT3, L2K, or F6A.

3. The T-cell antigen coupler of claim 2, wherein the CD 3-targeting single-chain antibody is UCHT1, and the nucleotide sequence of the CD 3-targeting single-chain antibody is shown in SEQ ID NO: shown at 10.

4. An expression vector comprising the T cell antigen coupler of claim 1 and a vector.

5. The expression vector of claim 4, wherein the vector is a lentiviral vector.

6. Use of a T-cell antigen coupler targeting CD19 according to claim 1, comprising: the T cell antigen coupler targeting CD19 is used as an anti-tumor drug.

7. The use of claim 6, wherein the tumor comprises: acute lymphocytic leukemia, chronic B-lymphocytic leukemia, non-Hodgkin's lymphoma, and multiple myeloma.